PDE3 cyclic nucleotide phosphodiesterases are used clinically to overcome a reduction in receptor- mediated cAMP generation in patients with heart failure. In the short term, PDE3 inhibitors have beneficial inotropic effects, but their long-term use has been associated with an increase in mortality that may be attributable to pro-apoptotic actions. Finding a way to attain the inotropic actions of PDE3 inhibition without the adverse long-term consequences would represent a significant therapeutic advance. In cardiac myocytes, PDE3 activity is found in intracellular compartments represented in cytosolic and microsomal fractions. Inotropic actions are likely to result from the inhibition of membrane-associated PDE3 activity and the consequent increase in cAMP-stimulated Ca2+ cycling, while pro-apoptotic changes in gene transcription are more likely to result from the inhibition of cytosolic PDE3 activity. Selective inhibition of membrane-associated activity might have inotropic actions without pro-apoptotic effects, but the three isoforms of PDE3 in cardiac myocytes - PDE3A1, PDE3A2 and PDE3A3 - are identical in their sensitivity to conventional catalytic-site inhibitors. Recent experiments, however, indicate that membrane-associated PDE3 isoforms integrate into multiprotein complexes with other membrane-associated proteins, providing a mechanism through which they can selectively regulate the phosphorylation of proteins involved in cAMP- mediated signaling in a specific compartment of cardiac myocytes. Blocking the interactions of PDE3 isoforms with these binding proteins may be a novel mechanism through which membrane-associated PDE3 activity can be targeted without inhibiting cytosolic activity. The availability of an agent that can selectively block the interactions of PDE3 isoforms with their membrane-associated binding proteins would allow us to test this hypothesis. With this goal in mind, our specific aims are: 1. To identify the proteins that interact with membrane-associated PDE3 isoforms in human myocardium: PDE3A-binding proteins in purified high-density microsomes from human left ventricular myocardium will be co-immunoprecipitated with anti-PDE3 antibodies and co-affinity purified with tagged recombinant PDE3 isoforms. PDE3A-binding proteins will be identified by Western blotting and mass spectrometry. Interactions with PDE3 isoforms will be confirmed by reverse co-immunoprecipitation in preparations from native tissue and co-transfected cells and by quantitation of protein-protein interactions by ELISA and surface plasmon resonance. 2. To identify the amino-acid sequences in these PDE3 isoforms that are involved in these interactions: Arrays of overlapping 25-mer peptides generated from the complete amino-acid sequences of PDE3A isoforms will be probed with the PDE3A-binding proteins identified in Aim 1 to determine the specific amino-acid sequences in PDE3A with which these proteins interact. 3. To determine whether peptides generated from these amino-acid sequences can be used as competitive inhibitors to disrupt these interactions: Synthetic peptides incorporating the PDE3A- derived amino-acid sequences identified in Aim 2 will be tested for their ability to inhibit protein-protein interactions and to block co-immunoprecipitation or co-affinity purification of PDE3A-binding proteins from cardiac microsomes. Achieving these aims will yield new insight into the mechanisms through which PDE3 isoforms regulate cAMP-mediated signaling in intracellular compartments of cardiac myocytes, and will provide us with prototypical agents that will be used in future experiments to test the feasibility of membrane-selective PDE3 inhibition as a therapeutic strategy for heart failure.